The present invention relates to methods for the selective increase of anticarcinogenic glucosinolate derivatives in Brassica species, and to Brassica species with enhanced levels of anticarcinogenic glucosinolate derivatives and in particular edible Brassica vegetables with elevated levels of the anticarcinogenic glucosinolate derivatives 4-methylsulfinylbutyl isothiocyanate and/or 3-methylsulfinylpropyl isothiocyanate. The present invention also provides methods for selection of genetic combinations of broccoli containing high levels of anticarcinogenic glucosinolate derivatives and methods to evaluate the anticarcinogenic properties of these genetic combinations. The invention further relates to compositions of matter comprising Brassica vegetables with concentrations of 4-methylsulfinylbutyl glucosinolate and/or 3-methylsulfinylpropyl glucosinolate between 10 and 100 xcexcmoles/g dry weight.
The present invention provides methods for the production of Brassica vegetables with elevated levels of specific glucosinolates and derivatives thereof. In particular the invention provides methods for the production and selection of Brassica vegetables with elevated levels of 3-methylsulfinylpropyl and/or 4-methylsulfinylbutyl glucosinolates. These glucosinolates are converted by the activity of the enzyme myrosinase into isothiocyanate derivatives which have been demonstrated to be potent inducers of phase II detoxification enzymes, elevated activity of which is associated with reduced susceptibility to the neoplastic effects of carcinogens. The invention provides genetic combinations which 1.) exhibit elevated levels of 4-methylsulfinylbutyl glucosinolate and/or 3-methylsulfinylpropyl glucosinolate and 2.) exhibit low activity of the GSL-ALK allele which encodes an activity capable of converting these glucosinolates into the alkenyl derivatives, which do not posses the anti-carcinogenic properties of the isothiocyanate derivatives of these glucosinolates and 3.) suitable myrosinase activity capable of producing isothiocyanate derivatives of said glucosinolates. Accordingly these genetic combinations provide elevated levels of specific glucosinolates, reduced production of alkenyl derivatives of these glucosinolates and favoured production of isothiocyanate derivatives of said glucosinolates. The invention further relates to the use of genetic markers to select the genetic combinations described above.
It is known that a diet high in vegetables is associated with a reduction in the risk of certain types of cancer and hence it is desirable to include a significant amount of vegetables in the human diet. The anticarcinogenic activity of vegetables has been associated with the presence of several classes of secondary metabolites. Evidence is growing that some of these secondary metabolites are involved in lowering the risk of certain types of cancer and hence are considered anticarcinogenic. Accordingly, enhancing the level of anticarcinogenic metabolites provides a useful strategy for the reduction of cancer risk, in complementation with dietary advice to increase the consumption of vegetables.
The precise mechanism by which vegetables provide a decreased risk of many types of cancer is not known with certainty, but there are many lines of evidence which support the involvement of vegetables in the prevention of cancer. In particular, the role of cruciferous vegetables in the prevention of cancer is widely supported through epidemiological studies and more recently biochemical studies. One class of secondary metabolites that is implicated in the beneficial effects of cruciferous vegetables is the isothiocyanate derivatives of certain glucosinolates. Four complementary pieces of evidence suggest that isothiocyanates derived from the hydrolysis of methylsulfinylalkyl glucosinolates found in crucifers may be important in the human diet in reducing the risk of cancer. (1.) Dietary provision of cruciferous vegetables protects rodents against chemically induced cancer (Wattenberg, L. W. (1985) Cancer Res. 45, 1-8.). (2.) Methylaulfinylalkyl isothiocyanates are known to be potent inducers of phase II detoxification enzymes in murine hepatoma Hepa 1c1c7 cells in culture (Zhang, Y., Talalay, P., Cho, C.-G., and Posner, G. H. (1992) Proc. Natl. Acad. Sci. USA 89, 2399-2403 and Tawfiq, N., Heaney, R. K., Plumb, J. A., Fenwick, G. R., Musk, S. R. R., and Williamson, G. (1995) Carcinogenesis 16, 1191-1194.), which are associated with reduced susceptibility of mammals and mammalian cell cultures to the toxic and neoplastic effects of carcinogens. (3.) Sulforaphane (4-methylsulfinylbutyl isothiocyanate) blocks the formation of mammary tumors in Sprague-Dawley rats treated with 9,10-dimethyl-1,2-benzanthracene (Zhang, Y., Kensler, T. W., Cho, C.-G., Posner, G. H., and Talalay, P. (1994) Proc. Natl. Acad. Sci. USA 91, 3147-3150.). (4.) Epidemiological studies show that people with high levels of vegetables in their diet are less susceptible to cancer (Block, G., Patterson, B., and Suber, A. (1992) Nutr. and Cancer 18, 1-19.). Thus the beneficial effects of a diet high in certain glucosinolates may included a reduction in the risk of cancer. However, it appears that only certain glucosinolates and more accurately, certain derivatives of specific glucosinolates may be primarily responsible for the beneficial effect.
There are numerous individual glucosinolates in cruciferous plants. Glucosinolates have a common glycone moiety and a variable aglycone side chain. The structure of the glucosinolate side chain varies in length and chemical composition.
Glucosinolates are formed by the action of a number of enzymes, encoded by a small number of glucosinolate biosynthetic alleles (GSL alleles). In the glucosinolate pathway, methionine is converted to homo-methionine and dihomo-methionine by the activity of the GSL-ELONG allele. Homo-methionine is eventually converted to 3-methylthiopropyl glucosinolate followed by conversion to 3-methylaulfinylpropyl glucosinolate by the activity of GSL-OXID allele and finally 2-propenyl glucosinolate by the activity of GSL-ALK allele. Dihomo-methionine is converted to 4-methylthiobutyl glucosinolate, then to 4 methylsulfinylbutyl glucosinolate by the activity of GSL-OXID allele, then to 3-butenyl glucosinolate by the activity of GSL-ALK allele and finally converted to 2-hydroxy-3-butenyl glucosinolate by the activity of GSL-OH allele.
In general, the 3-methylsulfinylpropyl glucosinolates and 4-methylthiobutyl glucosinolates produce non-volatile isothiocyanates and hence these particular glucosinolates contribute little to flavour. In contrast, the volatile alkenyl derivatives can contribute to flavour, both positively and negatively, dependant on the plant species and particular glucosinolate derivative.
In B. oleracea vegetables, glucosinolates have either a three or four carbon side chain. Glucosinolates can be hydrolysed by the action of myrosinase which is often induced upon tissue damage. Many vegetables have alkenyl (2-propenyl and 3-butenyl) glucosinolates which result in the production of volatile products upon hydrolysis through the action of myrosinase. Some vegetables contain a 2-hydroxy-3-butenyl glucosinolate called progoitrin. This glucosinolate produces an unstable isothiocyanate that spontaneously cyclizes to produce oxazolidone-2-thiones, which are undesirable in diets due to their goitrogenic properties. Isothiocyanates derived from alkenyl and hydroxyalkenyl glucosinolates can have both positive and negative effects on flavour.
Broccoli accumulates low levels of glucosinolates with 4-metylsulfinylbutyl and 3-methylsulfinylpropyl side chains since broccoli has a greatly reduced activity of the GSL-ALK allele, responsible for the conversion of glucosinolates into alkenyl derivatives. It is believed that the popularity of broccoli as a vegetable is due in part to the relatively modest contribution to taste made by 4-metylsultinylbutyl and 3-methylsulfinylpropyl glucosinolate derivatives in contrast to the strong flavour imparted by other glucosinolates, particularly the volatile derivatives of glucosinolates.
Thus, methods to increase the dietary amount of specific isothiocyanate derivatives of certain glucosinolates may provide vegetables with enhanced anticarcinogenic properties without altering the taste and/or palatability of the vegetable. However, the art does not provide a means to conveniently increase the levels of the specific glucosinolates in cruciferous vegetables. Moreover, the art does not provide a convenient means to assure that these glucosinolates are not converted to the alkenyl derivatives, but rather the isothiocyanate derivatives which have anticarcinogenic properties. Of the numerous glucosinolates that may be produced by Brassica vegetables, 4-methylsulfinylbutyl glucosinolate and 3-methylsulfinylpropyl glucosinolate have been identified as being the precursors to the most potent anticarcinogenic isothiocyanate derivatives. The art does not provide a convenient means to increase these specific glucosinolates in a specific fashion while preventing the formation of other glucosinolate or glucosinolate derivatives that may have undesirable flavour characteristics.
4-Methylsulfinylbutyl glucosinolate and 3-methylsulfinylpropyl glucosinolate glucosinolates are found in several cruciferous vegetables, but are most abundant in broccoli varieties (syn. calabrese: Brassica oleracea L. var. italica) which lack a functional allele at the GSL-ALK locus. The presence of a functional GSL-ALK allele converts these glucosinolates to their alkenyl homologues, which are poor inducers of phase II enzymes (Tawfiq, N., Heaney, R. K., Plumb, J. A., Fenwick, G. R., Musk, S. R. R., and Williamson, G. (1995) Carcinogenesis 16, 1191-1194.). Therefore the presence of a functional GSL-ALK allele precludes the possibility of producing a variety with high levels of these anticarcinogenic isothiocyanates since the glucosinolates will be converted to alkenyl derivatives. Additionally the production of isothiocyanates from glucosinolates requires the activity of the enzyme myrosinase. Hence enhanced production of these specific isothiocyanates depends on both the levels of glucosinolate precursors (which are influenced by the activity encoded by the GSL-ALK allele) and the levels or activity of myrosinase which produces the isothiocyanate derivatives of glucosinolates.
Accordingly a genetic combination which specifies the production of high levels of 4-methylsulfinylbutyl glucosinolate and/or 3-methylsulfinylpropyl glucosinolates is desirable, but the production of the anticarcinogenic isothiocyanate derivatives of these glucosinolates requires additional genetic combinations. Thus methods to achieve these genetic compositions provides novel compositions of matter not presently found in commercially grown cruciferous vegetables. The present invention recites methods for achieving these genetic combinations.
The levels of glucosinolates in commercially grown broccoli are relatively low compared to those found in salad crops such as rocket (Eruca sativa), which accumulates 4-methylthiobutyl glucosinolate, and watercress (Rorippa nasturtium-aquaticum) which accumulates phenethyl glucosinolate (Fenwick, G. R., Heaney, R. K., and Mullin, W. J. (1983) Crit. Rev. Food Sci. Nutr. 18, 123-201). Exposure to enhanced levels of 4-methylsulfinylbutyl glucosinolate and/or 3-methylsulfinylpropyl glucosinolate in broccoli would be expected to enhance the potency of induction of phase II enzymes when ingested. Thus broccoli with increased levels of the anticarcinogenic 4-methylsulfinylbutyl isothiocyanate and/or 3-methylsulfinylpropyl isothiocyanate would be a valuable addition to a diet that is designed to lower the risk of cancer. Additionally, such changes would be unlikely to lead to reduced palatability as methylsulfinylalkyl glucosinolates are non-volatile and have a relatively small contribution to flavour, in contrast to the majority of other isothiocyanates found in vegetables and salad crops (Fenwick, G. R., Heaney, R. K., and Mullin, W. J. (1983) Crit. Rev. Food Sci. Nutr. 18, 123-201). Thus altering the levels of these specific glucosinolates would not change the taste of the cruciferous vegetables which carry the genetic combinations encoding the trait.
Many wild members of the Brassica oleracea species complex (chromosome number, n=9) have high levels of individual aliphatic glucosinolates (Mithen, R., Lewis, B. G., and Fenwick, G. R. (1987) Phytochemistry 26, 1969-1973. and Giamoustaris, A. and Mithen, R. (1996) Theor. Appl. Genet. 93, 1006-1010.). Studies on the genetics of glucosinolates in these taxa has been instrumental in elucidating the genetic pathway for glucosinolate biosynthesis. It is evident that certain species in this taxa could be valuable in Brassica breeding programs designed to specifically enhance 4-methylsulfinylbutyl glucosinolate and/or 3-methylsulfinylpropyl glucosinolate and, by so doing, the anticarcinogenic potential of the plant. However, the art does not provide methods to increase the concentration of 4-methylsulfinylbutyl glucosinolate and/or 3-methylsulfinylpropyl glucosinolate through genetic combinations nor does it provide a convenient means by which the anticarcinogenic properties of the vegetables containing said genetic combinations can be assessed. The present invention provides these methods and genetic combinations.
Foremost amongst these are genetic combinations which incorporate the genes from members of the B. villosa-rupestris complex from Sicily, which possess a non-functional GSL-ALK allele, and may be the wild progenitors of cultivated broccoli. Thus the present invention utilizes wild relatives and progenitors of commercial broccoli as a source of the genes needed to derive a genetic combination capable of producing high levels of 4-methylsulfinylbutyl and/or 3-methylsulfinylpropyl glucosinolates and the genetic combination that favours the production of isothiocyanate derivatives of these glucosinolates rather than alkenyl derivatives.
4-Methylsulfinylbutyl isothiocyanate (also referred to as sulforaphane), derived from the corresponding glucosinolate found in some Brassica species, has previously been identified as a potent inducer of phase II detoxification enzymes (e.g. QR; quinone reductase [NADP(H):quinone-acceptor] oxidoreductase) in murine hepatoma Hepa 1c1c7 cells. Similarly, 3-methylsulfinylpropyl isothiocyanate is a strong inducer of phase II enzymes. Measurement of the induction of QR in murine hepatoma Hepa 1c1c7 cells provides a rapid and reliable indicator of the ability of vegetable extracts to induce phase II enzymes in mammalian cells (Prochaska, H. J., Santamaria, A. B., and Talalay, P. (1992) Proc. Natl. Acad. Sci. USA 89, 2394-2398.), and hence of putative anticarcinogenic activity. This assay has been used to assess the potential of synthetic isothiocyanates (Zhang, Y., Talalay, P., Cho, C.-G., and Posner, G. H. (1992) Proc. Natl. Acad. Sci. USA 89, 2399-2403 and Talalay, P., De Long, M. J., and Prochaska, H. J. (1988) Proc. Natl. Acad. Sci. USA 85, 8261-8265.), extracts from cruciferous vegetables (Tawfiq, N., Wanigatunga, S., Heaney, R. K., Musk, S. R. R., Williamson, G., and Fenwick, G. R. (1994) Exp. J. Cancer Prev. 3, 285-292.) and myrosinase-treated glucosinolates (Tawfiq, N., Heaney, R. K., Plumb, J. A., Fenwick, G. R., Musk, S. R. R., and Williamson, G. (1995) Carcinogenesis 16, 1191-1194). However, the glucosinolate/isothiocyanate content of the vegetable extracts has generally not been reported nor has the relative anticarcinogenic potential of various cruciferous vegetables been reported.
In the present invention, this assay has been used to determine the relationship between the ability to induce QR activity (anticarcinogenic potential) and the glucosinolate, content of three wild members of the B. oleracea complex, which have high levels of 3-methylthiopropyl (B. drepanensis) 3-methylsulfinylpropyl (B. villosa) and 2-propenyl (B. atlantica) glucosinolates respectively, when combined with commercial broccoli cultivars through conventional crosses and hybrids between the wild accessions and a commercial double haploid broccoli breeding lines. Accordingly methods and compositions have been derived which identify the genetic compositions required for the stable production of specific glucosinolates (e.g. of 4-methylsulfinylbutyl and/or 3-methylsulfinylpropyl glucosinolates) and the production of the corresponding isothiocyanate derivatives. These genetic combinations provide useful breeding lines for the production of commercial varieties of broccoli and other cruciferous vegetables that have 10-100 times the levels of these anticarcinogenic compounds than currently found in commercially grown varieties.
It has been found for example, that a ten-fold increase in the level of 4-methylsulfinylbutyl glucosinolate is obtained by crossing broccoli cultivars with selected wild taxa of the Brassica oleracea (chromosome number, n=9) complex. Similarly increases in levels of 3-methylsulfinylpropyl glucosinolate is observed. Tissue from these hybrids exhibited a 100-fold increase in the ability to induce quinone reductase in Hepa 1c1c7 cells over commercially grown broccoli cultivars due to both an increase in 4-methylsulfinylbutyl and/or 3-methylsulfinylpropyl glucosinolates and the increased conversion of 4-methylsulfinylbutyl glucosinolate to sulforaphane. Accordingly the invention provides methods and genetic compositions for the production of commercially valuable cruciferous vegetables containing high levels of anticarcinogenic secondary metabolites. The invention further contemplates the development of broccoli breeding lines with enhanced anticarcinogenic activity.
The selection of breeding lines with high levels of anticarcinogenic compounds is further facilitated by the use of molecular markers to establish the chromosomal location of the glucosinolate biosynthetic genes and to assist in selection of backcross lines which contain the genetic composition of greatest utility for the purposes of enhancing the levels of anticarcinogenic compounds in broccoli.
The present invention provides methods to increase levels of 4-methylsulfinylbutyl and/or 3-methylsulfinylpropyl glucosinolates in Brassica vegetables by genetic means. These means include crossing of wild Brassica species to broccoli species, selection of lines with elevated 4-methylaulfinylbutyl and/or 3-methylaulfinylpropyl glucosinolates and evaluating the anticarcinogenic properties of said genetic combinations by measuring the potency of plant cell extracts to induce phase II enzymes. RPLP markers can be used to select lines in crosses which have a high proportion of broccoli genetic background and to establish the position of the relevant glucosinolate biosynthesis genes on the map of the Brassica genome.
Hybrids between commercial broccoli cultivars and the two wild species B. villosa and B. drepanensis are fully fertile and backcross populations are made. Broccoli lines with enhanced levels of 4-methylsulfinylbutyl and/or 3-methylsulfinylpropyl glucosinolates and associated anticarcinogenic activity are developed from these populations. The efficiency of the development of these lines is considerably enhanced by the availability of molecular markers to select for both glucosinolate content and the desired genetic background.